Optimization of the microalgae culture conditions could significantly reduce the production costs of microalgae-derived biodiesel. In the current study, a new process of adding different forms using the multiple small-dose method was employed. The effects of different forms of nitrogen (NaNO3, NH4Cl, and CH4N2O) and their concentrations (0.1, 0.5, 1, and 2 mg L−1) on the growth and lipid production of Scenedesmus obliquus were studied. Algae density and lipid production increased with increasing nitrogen concentration for all different forms of nitrogen except NH4Cl. The Scenedesmus obliquus growth was promoted by adding NaNO3 and CH4N2O, but was inhibited by adding NH4Cl. Adding 2 mg N L−1 of CH4N2O daily yielded the highest cell density (1.7 × 107 cells mL−1) and lipid production (242.4 mg L−1). These conditions can thus maintain the biomass of Scenedesmus obliquus, increase its lipid accumulation, and decrease the costs of biodiesel production.